Accepted Manuscript
Title: Evaluating misoprostol content in pregnant women with
hourly oral administration during labor induction by
microElution solid phase extraction combined with liquid
chromatography tandem mass spectrometry
Author: Cheng-Han Hung Shi-Yann Cheng Tzu-Min Chan
Maw-Rong Lee
PII:
DOI:
Reference:
S1570-0232(15)30081-7
http://dx.doi.org/doi:10.1016/j.jchromb.2015.07.012
CHROMB 19509
To appear in:
Journal of Chromatography B
Received date:
Revised date:
Accepted date:
25-4-2015
1-7-2015
5-7-2015
Please cite this article as: Cheng-Han Hung, Shi-Yann Cheng, Tzu-Min Chan, MawRong Lee, Evaluating misoprostol content in pregnant women with hourly oral
administration during labor induction by microElution solid phase extraction combined
with liquid chromatography tandem mass spectrometry, Journal of Chromatography B
http://dx.doi.org/10.1016/j.jchromb.2015.07.012
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Evaluating misoprostol content in pregnant women with hourly oral
administration during labor induction by microElution solid phase extraction
combined with liquid chromatography tandem mass spectrometry
Cheng-Han Hung a, Shi-Yann Cheng b,c,d, Tzu-Min Chan d, Maw-Rong Lee a,*
a
Department of Chemistry, National Chung Hsing University, Taichung 40227,
Taiwan, R.O.C.
b
School of Medicine, China Medical University, Taichung 40402, Taiwan, ROC
c
Department of Obstetrics and Gynecology, and dDepartment of Medical Education
and Research, China Medical University Beigang Hospital, Yunlin 65152, Taiwan,
ROC
* Corresponding author. Tel: +886 422851716; Fax: +886 422862547.
E-mail address: mrlee@dragon.nchu.edu.tw
Abstract:
Misoprostol is a widely used alternative of prostaglandin for labor induction.
Based on previous studies, we envision that small and frequent oral dosage of
misoprostol is an effective method for labor induction. To monitor the misoprostol
content during labor induction, a rapid, sensitive, and selective microElution solid
phase extraction (μElution SPE) combined with liquid chromatography tandem mass
spectrometry (LC-MS/MS) was developed. Using μElution SPE could minimize the
sample consumption and elution volume in order to maximize the sample enrichment and
throughput. The misoprostol acid, a metabolite of misoprostol, was gradient
separated in a Bidentate C18 column, then quantified by highly-selective reaction
monitoring (H-SRM) in a total run time of 6 min. The developed method was
1
optimized and validated in human plasma, and showed linear range of 0.01-10 ng/mL.
The limit of detection (LOD) was 0.001 ng/mL. The recovery ranged from 89.0 to
96.0%, and no significant matrix effect or carryover was observed. The precision,
accuracy and stability were met with the criteria of U.S. FDA guidance. The developed
method was successfully applied to evaluate misoprostol concentration during labor
induction in pregnant women. The concentration-time profiles approves that hourly
oral administration of misoprostol is a safe and effective method without drug
accumulation for labor induction.
Keywords: Labor induction, Misoprostol acid, Liquid chromatography tandem mass
spectrometry, highly-selective reaction monitoring, microElution solid phase extraction
1. Introduction
Labor induction is widely used over the world because continuation of
pregnancy is harmful to a mother and her fetus. In World Health Organization (WHO)
Global Survey, the delivery involved labor induction was as high as 35.5% in Asian
countries [1]. Prostaglandins are the most effective choice for helping labor induction.
The main disadvantages of prostaglandins are that they are expensive and sensitive to
temperature changes. The affordable alternative, misoprostol, is a prostaglandin E1
analogue, which has some advantages, such as high stability at room temperature, cheap,
and could be given in several routes [2,3]. It is a safe, effective, and promising method
for both nulliparous and multiparous women, and it is more efficient with oral
administration than vaginal administration [4,5]. From the results of clinical trials in
previous studies, we envision that using small and frequent oral doses of misoprostol is
an effective method for labor induction. To approve the hypothesis and establish a
2
reliable protocol for labor induction, it is important to monitor the misoprostol
concentration during the process of hourly administration.
Misoprostol was readily metabolized to its pharmacologically active form,
misoprostol acid (MPA), after five minutes of oral administration. The peak
concentration of misoprostol acid was achieved in 12 ± 3 min, and its half-life was 20
to 40 min, then declined rapidly thereafter [6]. Independent of the route of
administration, the therapeutic dose does not usually exceed 0.8 mg per day. To assess
safety of misoprostol, the total dosage and side effects were considered. The safety of
misoprostol has been documented in dosage up to 1.6 mg per day [7] and the side effects
of the drug are diarrhea, pyrexia and shivering. But even higher dosage of misoprostol
applied, only mild side effects have been observed. Several published case reports
[8,9,10] demonstrate that overdosing of misoprostol with single dosage more than 3.0
mg resulted in hyperthermia, hypoxia and rhabdomyolysis. In previous clinical pilot study
[11], the maximum total dosage was 9.6 mg in nulliparous woman who only
experienced the diarrhea and it is the most common side effect (50%) in the study.
Because of recommended therapeutic dose is low, the highest concentration of the
bioactive metabolite is very low [12]. Therefore, an analytical method with high
sensitivity and selectivity is needed for determining the concentration of misoprostol
acid in plasma.
Determination of misoprostol acid in biological matrices is usually accomplished
by several analytical methods, such as radioimmunoassys [6], gas chromatographynegative ionization chemical ionization tandem mass spectrometry (GC-NICI-MS/MS)
[13,14], and liquid chromatography-tandem mass spectrometry (LC-MS/MS)
[12,15,16]. The drawback of GC-NICI-MS is that it requires complicated
derivatization procedures, long chromatographic time, and sacrificed sensitivity with
3
syn and anti isomer. Solid phase extraction (SPE) is one of the widely used
preparation methods for LC-MS/MS, which could combine the cleanup and
enrichment at the same time. Usually, SPE requires the large amount of sample and
elution solvent, from milliliter to liter range, and needs to convert the eluent into the
suitable solvent in order to achieve better LC separation. These steps are not only
time-consuming but also cause experiment errors. microElution solid phase extraction
(μElution SPE) has the unique design to allow the loading of 10-750 μL of sample and
eluting of ultra-low elution solvent, which is 25 μL [17-21]. These properties help to
avoid using large amount of sample, eliminate the evaporation and reconstruction step,
hence increasing the sample throughput. The sorbent used in this study, Oasis HLB
(Hydrophilic-Lipophilic Balance), is a divinylbenzene/N-vinylpyrrolidone polymer that
has both lipophilic and hydrophilic groups and exhibit excellent wetting properties.
Therefore, HLB could give maximum extraction efficiency for MPA in plasma sample
without the loss of recovery or breakthrough problem, which is the major drawback of
silica-C18 sorbent.
Here, we designed a simple, rapid, and reliable method for evaluating misoprostol
content in pregnant women during labor induction in order to prove the proposed
hypothesis. To achieve the purpose, the microElution solid phase extraction combined
with LC-MS/MS is developed to detect trace misoprostol acid in human plasma
sample and validated to meet the criteria of the U.S. FDA guidance.
2. Experimental
2.1 Materials, reagents and chemicals
Misoprostol acid (MPA) and misoprostol acid-d5 (MPA-d5), as the internal
standard (IS), were purchased from Cayman Chemicals (Ann Arbor, MI, USA).
4
Ultrapure water (>18 MΩ) was purified with a Milli-Q system (Millipore Simplicity®,
Millipore, France). The quality of the solvents and chemicals used during this study were
HPLC grade or better. The stock solution of MPA (1 mg/mL) and MPA-d5 (50 μg/mL)
were prepared in methanol and stored in the refrigerator at -30 ◦C. Standard working
solutions were prepared daily by mixing stock solutions and methanol-water (1:1, v/v)
to the required concentrations. MicroElution 96-well SPE (μElution SPE) with Oasis
HLB sorbent (2 mg) were obtained from Waters (Milford, MA, USA).
Human plasma samples and drug-free plasma were obtained from China
Medical University Beigang Hospital (Yunlin, Taiwan). These samples used in this
pilot study were collected from three healthy pregnant women (27.5 ± 4.5 year-old)
who did not have a disease of the heart, liver, and kidney and an allergy to
misoprostol. No analgesic agent was used during labor induction and the common side
effect of diarrhea happened to one of three pregnant women who relieved the symptom
easily by anti-diarrhea agent. The formal ethic approval (IRB#DMR99-IRB-242,
approved on 09 Dec. 2010) was obtained from the institutional review board of China
Medical University Hospital, and volunteers provided with informed written consents.
For the evaluation of concentration of misoprostol during labor induction, the subjects
received a tablet of 200 μg misoprostol (Cytotec®, Pfizer Inc., Taiwan) by hourly oral
administration more than 8 hours. Human plasma samples were collected at 0h, 0.25h,
0.5h, 1h, 2h, 4h, 6h, and 8h in the clean K3EDTA-treated polypropylene tubes. After
centrifuging, the samples were stored in the refrigerator at -30 ◦C until analysis. Drugfree plasma were used for method development and validation.
2.2 Instrumentation
The Thermo Scientific Accela LC system and autosampler (Thermo Scientific,
5
San Jose, CA, USA) were used for chromatographic analysis. The gradient separation
was performed by Cogent Bidentate C18 column (100 mm × 2.1 mm i.d., 4 μm,
MicroSolv Technology, Eatontown, NJ, USA) with Bidentate C18 guard column (20
mm × 2 mm i.d.) at 30 ◦C and flow rate of 0.4 mL/min. The mobile phases were water
and acetonitrile. Gradient separation began at 40% acetonitrile, isocratic for 1 min,
then increased to 95% acetonitrile within 0.5 min, isocratic for 2 min. Afterward, the
conditions returned to 40% acetonitrile and equilibrated for 2 min, resulting a total run
time of 6 min. An injection volume of 5 μL was used.
The TSQ Quantum Ultra EMR triple quadrupole mass spectrometer (Thermo
Scientific, San Jose, CA, USA) was equipped with heated electrospray ionization (HESI). The MS analysis was operated in negative ionization mode with H-ESI and
quantified by using highly-selective reaction monitoring (H-SRM). The tuning
parameters were optimized for MPA by using post-column T infusion method, which
was infusing 10 μg/mL MPA through syringe pump at LC flow rate of 0.4 mL/min.
The optimized H-ESI voltage and temperature were set at 4.5 kV and 300 ◦C,
respectively. Sheath gas was set at 20 arbitrary unit and Aux gas at 10 arbitrary unit. In
H-SRM, argon was used as the collision gas at the pressure of 1.5 mTorr with
optimized collision energy of 20 eV for both MPA and IS. The mass transitions used
were m/z 367 > 249 for MPA and m/z 372 > 249 for MPA-d5. The resolution (FWHM,
full width at half-maximum) of Q1 and Q3 were operated at 0.4 and 0.7, respectively.
Raw data was acquired and processed by using Xcalibur 2.0.7 (Thermo Scientific).
2.3 Sample preparation
An aliquot of 200 μL sample containing IS solution (40 μL) was diluted with
200 μL of 0.1% formic acid, and then centrifuged at 14,000 rpm for 10 min. The
6
supernatant was transferred to another clean Eppendorf tube. The μElution SPE plate
was conditioned sequentially with 0.5 mL MeOH, 0.5 mL water and 0.5 mL 0.5%
acetic acid. An aliquot of 300 μL supernatant was loaded into μElution plate, and then
washed with 0.5 mL 0.5% acetic acid, 0.5 mL acetonitrile-water-acetic acid
(100:900:0.5, v/v/v), and 0.5 mL water. After that, the elution step was done with 50
μL of acetonitrile-water (50:50, v/v). The eluent was directly analyzed by LC-MS/MS.
2.4 Method validation and real sample analysis
The method was validated to meet the criteria of the U.S. FDA guidance,
Guidance for Industry: Bioanalytical Method Validation [22]. To evaluate method
performance, such as calibration curve, accuracy, precision, recovery, matrix effect,
and stability was systematically studied with triplicate analysis. The calibration curve
was established by blank sample, zero sample, and six non-zero sample, ranging from
0.01 to 10 ng/mL. The limit of quantification (LOQ) was defined as the lowest
concentration on the calibration curve, and both precision and accuracy should be less
than 20%. The limit of detection (LOD) was calculated by 3×SD/slope. The QC
samples were consisted of 3-level concentrations, 0.02, 0.5, and 10 ng/mL and applied
to evaluate the recovery, accuracy, matrix effect, intra-day and inter-day precision,
freeze/thaw stability, and short-term stability.
To determine the extraction recoveries of μElution SPE, the peak areas of
extracted MPA spiked sample and standard solution of the equivalence concentration
were compared. The accuracy and precision were calculated on the analysis of samples
containing known amounts of the MPA. The precision was subdivided to intra- and
inter-day precision and represented by relative standard deviation (%RSD). The
accuracy and precision were required to be within 15%. For evaluating the ion
7
suppression or ion enhancement phenomenon, the matrix effect (ME) was calculated
on peak areas of MPA spiked post-extraction sample (A) and standard solution of
equivalence concentration (B). The ratio (A/B × 100) within 80-120% indicates no
significant matrix effect. A ratio of >120% indicates ion enhancement, and a ratio of
<80% indicates ion suppression. The freeze/thaw stability of misoprostol acid in the
sample was evaluated after three complete freeze/thaw cycles between the storage
temperature and room temperature. For the evaluation of short-term stability, samples
were exposed to room temperature for 4 hours.
The validated method was applied to the analysis of plasma samples for clinical
monitoring the
misoprostol concentration during the labor induction. The
concentration of sample was quantified by calibration curve and represented as real
amount in the sample.
3. Results and Discussion
3.1 Method development
3.1.1 Optimization of LC-MS/MS method
For achieving the maximum sensitivity, selectivity and high throughput, the
chromatographic separation is critical. There are three main factors that affect the
chromatographic efficiency: column specification (length, diameter, particle size and
stationary phase), mobile phase constitution, and separation method (isocratic or
gradient). In this study, Bidentate C18 column was employed. Unlike ordinary silicabased column, Bidentate C18 was built on silica hydride material (Si-H) which replaces
95% of the silanols group on the stationary phase [23,24]. This property could avoid
the unwanted interaction between analyte and stationary group for improving peak
shape. In previous literature, Pesek and co-workers [23] investigated retention
8
behavior of silica hydride-based column. The result showed that peptide mixtures were
baseline separated under isocratic separation, and the chromatographic time reduced
from 45 min to 5 min by using Bidentate C18 column. According to these benefits,
Bidentate C18 column has the most suitable stationary phase for determining the MPA
concentration in plasma sample. Mobile phase composition is another main factor to
affect chromatographic efficiency and sensitivity, especially for LC-MS. Acetonitrile is a
stronger eluent in reversed phase chromatography and also is a better spray solvent for
ESI ionization. These properties of acetonitrile could help to achieve the purpose of
reducing chromatographic time and enhancing ionization efficiency. Schmidt and coworkers [25] systematically studied the chromatographic conditions of LC-ESI- MS/MS
for determining the prostaglandin E2 and D2 that have the similar structure of misoprostol
acid. The result showed that using non-acidic mobile phase and gradient separation
could achieve best signal-to-noise ratio, in a two-fold improvement. Hence, Gradient
separation with acetonitrile and water was chosen to enhance the selectivity and reduce
the chromatographic time.
In MS analysis, misoprostol acid is a polar compound, which has a carboxyl
group and can easily lose the hydrogen to generate a negative ion. Therefore, MPA
could produce intense negative ion by using ESI negative ionization mode. Based on
direct infusion analysis, MPA and IS formed the deprotonated molecule ion ([M-H]-)
at m/z 367 and 372, respectively. In MS/MS analysis, both MPA and IS were obtained
the intense fragment ion at m/z 249, [M-H-H2O-C6H12O]- for MPA and [M-H-H2OC6H7D5O]- for IS. To increase ionization efficiency, H-ESI with heated auxiliary gas
was applied for desolvation of sample solution. To improve the selectivity and
reliability of the purposed method, H-SRM was utilized to enhance resolution and
reduce matrix interference. The advantages of H-SRM were well described in previous
9
articles [26–29]. Jemal and a co-worker [28] described that using H-SRM for
determining the nefazodone in human plasma could produce cleaner chromatograms,
which means higher signal-to-noise ratio and potentially better effective sensitivity. For
enhancing resolution and reducing matrix interference, H-SRM was utilized and set to
0.4 FWHM at Q1, and to 0.7 FWHM at Q3. Additionally, the definition of resolving
power is m/Δm, hence the resolution increased approximately twofold while applying
H-SRM for determining the misoprostol content. Optimal collision energy in H-SRM
was evaluated by automatic instrument adjustment. Based on the result, optimal collision
energy for MPA and IS was 20 eV.
3.1.2 μElution SPE
For LC-MS/MS analysis, sample preparation is extremely important. An
efficient extraction could reduce matrix interference for LC separation and improve
ESI ionization efficiency. The extraction method applied to HLB μElution SPE was
modified from the previous article [30] and optimized for MPA in spiked plasma.
The spiked sample was first diluted with formic acid solution, which converts
MPA from ionic form into neutral form, and was also the process for protein
precipitation. The concentration of formic acid was evaluated from 0.05 to 0.5 %. No
significant effect was observed. Then 0.1 % formic acid was chosen because of the better
precision. To optimize the extraction efficiency for MPA, the loading volume and
elution volume in HLB μElution SPE was systematically studied. The loading volume
was evaluated from 0.1 to 0.3 mL. The result showed improved extraction efficiency
with increasing the loaded sample volume. Sample volume higher than
0.3mL was not studied because of the low sorbent amount, which was 2 mg. Too
much loaded sample would possibly cause clogging problem, which reduces the sample
10
transfer and also lengthens the extraction time. To achieve the maximum elution
efficiency and eliminate the reconstitution step, 50 μL of an acetonitrile-based elution
solvent was chosen. Elution by pure acetonitrile caused peak broadening and retention
time shift. Therefore, water-acetonitrile (50:50, v/v) and two-step elution by water and
acetonitrile were compared. The water-acetonitrile solution gave slightly better elution
efficiency by about 13%, compared to the two-step elution. In addition, adding acetic
acid in the elution solvent (0 to 1%) did not improve or reduce the elution efficiency.
To confirm whether the elution process was complete or not, the HLB μElution SPE
was eluted twice with optimized elution solvent. The result showed that 90% of the
analyte was eluted in the first elution.
3.2 Method validation
Mass ion chromatograms of MPA and IS in a zero sample, a spiked plasma
sample and a plasma sample are shown in Fig. 1. No interfering peaks from
endogenous compounds or matrix were observed. The calibration curve was prepared
and analyzed as described previously. The coefficient of determination (R2) was higher
than 0.9983 over a concentration range of 0.01-10 ng/mL. The LOQ was 0.01 ng/mL
and LOD was 0.001 ng/mL. The validation results of plasma sample are summarized in
Table 1. The extraction recoveries of the HLB μElution SPE ranged from 89.0 to
96.0% in plasma sample. The intra-day precision ranged from 3.1 to 7.9%, and the
accuracy ranged from 98.3 to 102.1%. The inter-day precision ranged from 5.8 to
9.0%. The matrix effect was evaluated to understand the matrices interference, such as
ion suppression or ion enhancement. The results indicate that there were no significant
matrix effects in the plasma sample. After evaluating the short-term stability and three
freeze/thaw cycles, the concentration of MPA in matrices demonstrated acceptable
11
11
stability in three QC levels.
Comparing with the existing methods, the advantages of the proposed method are
lower sample consumption and simplified sample preparation steps, which do not need
reconstitution and derivatization steps. In contrast, the same level of LOD and LOQ
with the previous method is the one flaw of the proposed method.
3.3 Clinical monitoring for labor induction
The purposed method was applied to the analysis of plasma samples obtained
from healthy pregnant women as described previously. The concentration-time profiles
are depicted in Fig. 2. The concentration of MPA in plasma reaches the maximum (Cmax)
after 0.25-0.5 h and then decreases to the minimum after 1 h. This is because misoprostol
is easily and quickly metabolized into misoprostol acid. After 1 h, the concentration of
MPA increases slightly in 2-4 h, then reaching a fixed concentration if we were hourly
giving dosage of misoprostol. To make sure the potency of misoprostol during
labor induction process, it is important to keep the concentration of MPA reach the
constant. In previous literatures, Zou et al. [15] reported the Cmax was
857 ± 600 pg/mL in plasma for healthy non-pregnant women with single oral
administration of 0.6 mg of misoprostol. Abdel-Aleem et al. [31] described the Cmax
was 344.6 ± 268.9 pg/mL in plasma for postpartum women with single oral
administration of 0.6 mg of misoprostol. Khan et al. [32] reported Cmax was 327.9 ±
102.9 pg/mL in serum for pregnant women with oral administration of 0.6 mg of
misoprostol. Although the Cmax in this study is slightly smaller, the results still had in
good agreement with previous literatures. Again, we envision that using small and hourly
doses of misoprostol is a safe and effective method for labor induction. This
concentration-time profile proves the proposed hypothesis.
12
12
4. Conclusions
In this study, a μElution SPE combined with LC-MS/MS was developed and
validated for clinical monitoring on the misoprostol concentration in labor induction.
The advantage of μElution SPE is low sample consumption, eliminating reconstruction
step, and also less processing time in multi-sample treatment. In MS/MS analysis, HSRM reduces the matrix interference and the sensitivity is not sacrificed. The
developed method provided good precision and accuracy in the linear range of 0.01-10
ng/mL, using 200 μL of plasma sample, and the LOD was 0.001 ng/mL. This method
was validated in human plasma and successfully applied to estimate the misoprostol
concentration of pregnant women during labor induction. The result demonstrates that
hourly oral administration could keep the potency of misoprostol during labor induction.
Acknowledgement
The author would like to thanks the National Science Council of Taiwan for
supporting this research under contract no. of NSC98-2113-M-005-015-MY3.
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Figure captions:
Fig. 1. Mass ion chromatograms of MPA by using μElution SPE-LC-MS/MS. (a) zero
sample; (b) spiked 0.02 ng/mL of MPA in drug-free plasma; (c) plasma sample of subject
A, after 6 h of oral administration of misoprostol. The retention time of both MPA and
IS is 2.1 min.
Fig. 2. Concentration-time profile of MPA in human plasma after receiving a tablet of
200 μg misoprostol until 8 hours.
Table 1. Validation data of μElution SPE-LC-MS/MS for the analysis of misoprostol
acid in human plasmaa.
ng/mL
Recovery
Accuracy
Matrix effect
Intra-day precision
Inter-day precision
Freeze-and-th
(RSDb)
%
(RSD)
102.1
% (RSD)
95.0 (6.2)
(%, RSD)
7.9
(%, RSD)
9.0
ng/mL (SDc)
0.020 (0.002)
97.6 (5.2)
5.4
7.5
0.508 (0.039)
104.5 (2)
3.1
5.8
10.167 (0.682
0.02
%
89.0 (6.3)
0.5
94.1 (4.5)
10
96.0 (2.9)
(7.8)
101.2
(5.3)
98.3 (3.6)
a.
the experiments were conducted with triplicate analysis (n=3)
b.
RSD: Relative standard deviation
c.
SD: Standard deviation
Fig. 1
16
16
Fig. 2
17
18